Hitherto, a historical record of a physical condition has not been obtainable unless an indicator of the condition or disorder has been measured concurrently with the progression of the condition or disorder.
Red blood cells contain hemoglobin, a complex molecule involved in the transport of oxygen and carbon dioxide in the blood. They continuously enter into the circulation from the bone marrow, where they are made. The red blood cells first entering the blood stream are termed reticulocytes, and after the "reticulum" of nucleic acid material is eliminated from the reticulocytes, in the first two days of circulation, the red blood cells continue to circulate, as mature cells, for 120 days in normal people. After this time the red blood cells are eliminated from circulation and from the body.
It is well known that during their lifetime, red blood cells are bathed in fluid plasma with a continuously changing chemical composition. Some components of the plasma also move freely into and out of the red blood cell through the cell membrane. While in the red blood cell, some of these components exert an influence on the constituent molecules of the red blood cells, particularly on the hemoglobin molecule.
The hemoglobin (Hb) molecule consists of heme (a pigment) and globin (a protein). The heme contains iron in the ferrous state. The protein moiety is formed by two alpha-chains (141 amino acids) and two beta chains (146 A.A.). Normal hemoglobin is called hemoglobin A (HbA). Over 100 amino acid sequence variants are known.
A variety of chemically modified forms of hemoglobin are known. O.sub.2 combines rapidly and reversibly with hemoglobin to form oxyhemoglobin. When the iron of the hemoglobin is oxidized to the ferric form, methemoglobin is formed. This change is reversed by DPNH and methemoglobin reductase. Hb is irreversibly converted to sulfhemoglobin by certain drugs. Carboxyhemoglobin is made when carbon monoxide binds to hemoglobin. Cyanohemoglobin is the reaction product of Hb and cyanide ion.
Glycosylated hemoglobins are known. Ashby, et al., Diabetic Medicine 2:83-87 (1985); Mortensen, Danish Medical Bulletin, 36:369-328 (1985); Howard, et al., Acta Paediatr. Scand. 70:695-698 (1981); Mortensen, J. Chromatogr. 182:325-33 (1980). Glycohemoglobins retain the original function of hemoglobin, the transport of oxygen. The amount of glycohemoglobin formed is directly proportional to the concentration of glucose in circulating plasma which surrounds the red blood cells, and the time of exposure.
The proportion of glycohemoglobin to hemoglobin in a blood sample may be measured, and this measurement is performed as a chemical test, the general concept being the subject of U.S. Pat. Nos. 4,399,227, 4,448,888, 4,438,204, 4,372,747, and 4,465,774. In these tests, the hemoglobin and glycohemoglobin from cells of different ages has been mixed indiscriminately.
The five most prominent glycosylated hemoglobins are collectively designated HbA.sub.1. They are HbA.sub.1a1 (0.2%), HbA.sub.1a2 (0.2%), HbA.sub.1b (0.5%), HbA.sub.1c (4-6%) and HbA.sub.1d (0.2-0.6%). HbA and HbA.sub.1 combined make up about 97% of the total hemoglobin content.
Glycohemoglobin measurements have attracted attention as a possible indicator of dietary compliance by diabetics, see Howard, et al., Acta Pediatr. Scand., 70:695-98 (1981).
Hoberman, U.S. Pat. No. 4,463,098, provides for measuring the average historical record of alcohol consumption in a patient by recording the concentration of a hemoglobin molecule altered by reaction with a modified sugar, 5-deoxy-D-xylulose-1-phosphate (DXP) to form "DXP-hemoglobin". The sugar molecule becomes trapped on the hemoglobin molecule due to the indirect action of alcohol on the normal breakdown mechanism of sugar in the red blood cells. The measurements of the Hoberman patent are not made on a cell-by-cell basis, and they tend to average the day-to-day changes in alcohol level.
It is generally accepted that erythrocytes may be separated according to age on the basis of relative densities. Van Gastel, 1968-RVI, Methods for Studying the In Vivo Aging of Red Cells, 1-32 (Blood Information Service); Schulman, Biochim. Biophys. Acta, 148:251-55 (1967); Leif and Vinograd, Biochemistry, 51:520-28 (1964). This is disputed, however, by Mortenson, Danish Med. Bull., 32:309,320 (December 1985).
A variety of biochemical changes are believed to be associated with erythrocyte senescence. Phytrakul, M.S. Thesis, Age-Related Changes in Red Cell Activities of Glycolytic Enzymes, Reduced Glutathione, and Hemoglobin Denaturation, 1-85 (University of Oregon Health Sciences Center, Portland, Ore.: 1976); Keitel, et al., Blood 10:370-76 (1955); Bernstein, J. Clin. Invest., 38:1572-86 (1959); Edwards and Rigas, J. Clin. Invest., 46:1579-88 (1967).
Polychronakos, et al., J. Clin. Endocrin. Metab., 55:290 (1982) discovered that most if not all of the difference in insulin binding seen between the RBCs of the adult and the newborn is attributable to a greater preponderance of younger RBCs in the latter. Earlier studies established that reticulocytes bound about twice as much insulin as the oldest cells.
Fitzgibbons, et al., J. Clin. Investig., 58:820-824 found that both normal and diabetic erythrocytes contained greater amounts of hemoglobin HbA.sub.1a+b and Hb A.sub.1 c in the older cells. Likewise, Elseweidy, et al., J. Lab. Clin. Med., 102:628 (1981) felt that there is a red cell age-related increase in glyco Hb. On the other hand, Mortensen, Danish Med. Bull., 32:309, 320 (December 1985) reported that the differences in HbA.sub.1 c content between "young" (slow-sedimenting) and "old" (fast-sedimenting) cells were not statistically significant.
There is evidence to the effect that during the lifespan of the RBC, HbA is slowly and irreversibly glycosylated. Maney, et al., Blood, 46:1051 (1975) (abstract); Ashby, et al., (1985), supra; but see Mortensen (1985), supra. If this occurs in a mathematically predictable manner, then the ratio of glyco HbA to HbA is indicative of the age of the RBCs and may be used to detect age-related changes in other metabolites.
Stillman, U.S. Pat. No. 3,864,571, describes a fluorescence-activated cell counter which will separately count lymphocytes, polymorphonuclear neutrophils, eosinophils, monocytes, basophils, platelets, and reticulocytes, based on differential staining of their nuclei and cytoplasm. Wheeless Jr., U.S. Pat. No. 3,497,690 distinguishes normal cells from carcinogenic cells by nucleic acid content, as determined by a fluorochrome dye. Kamentsky, U.S. Pat. No. 3,413,464 relates to methods of enhancing this distinction. Staining is also employed by Groner, U.S. Pat. No. 3,740,143. See also U.S. Pat. Nos. 2,875,666, Parker; Fulwyler, 3,893,767; Adams, 3,883,247; Kamentsky, 3,662,176; Elkin, 3,661,460; Ehrlich, 3,699,336; Tyrer, 4,172,227; Bouton, 3,873,974; Miller, 3,827,804; and Miller, 3,832,687.
Fulwyler, U.S. Pat. No. 4,499,052 distinguishes cells with different relative receptivities for two different labeling agents by the ratio of the two labeling agents bound to the cell (e.g., fluorescein and rhodamine labeled particles).
Rogers, U.S. Pat. No. 4,416,778 described a method of preparing neocyte-enriched blood for use in blood transfusions. Neocytes (young RBCs) were centrifugally separated from gerocytes (old RBCs) based on their difference in density.